Id-1 promotes angiogenesis in hepatocellular carcinoma through HIF-1α-mediated VEGF upregulation

Abstract

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly vascularized tumor associated with poor prognosis. Angiogenesis plays a critical role in rapid growth and metastasis of HCC. Vascular endothelial growth factor (VEGF) is one of the most important angiogenic factors in HCC. A better understanding of the regulatory mechanisms of VEGF expression in HCC may reveal novel targets for treatment of this cancer. Recently, Id-1 (inhibitor of differentiation/DNA synthesis) has been suggested to play a role in cancer angiogenesis but the molecular mechanism remains unknown. Given the central role of VEGF in cancer angiogenesis, we performed a study to elucidate whether overexpression of Id-1 promotes angiogenesis in HCC by interacting with VEGF. METHODOLOGY: In this study, we examined the expression of Id-1 and its relationship with expression of VEGF in HCC by performing immunohistochemistry on HCC tissue microarray. The role of Id-1 in regulating angiogenesis in HCC was evaluated in vitro by ectopic Id-1 transfection, and the effect of inhibition of Id-1 expression on angiogenesis and tumor growth of HCC was studied in vitro and in vivo. RESULTS: Id-1over-expression correlated with HCC metastasis (p<0.001), and its expression significantly correlated with VEGF expression (p<0.001, r=0.47) by tissue microarray. Ectopic transfection of Id-1 into HCC cell lines induced VEGF secretion, and the culture medium of the Id-1 transfectants could induce morphological change and proliferation of human umbilical vein endothelial cells. Furthermore, the angiogenic effect of the culture medium of the Id-1 transfectants was reversed by treatment with an Flk-1 inhibitor, SU1498, or with a VEGF neutralizing antibody. Id-1 induced transcriptional activation of VEGF by upregulating its promoter harboring hypoxia inducible factor-1α (HIF-1α) binding site. Id-1 was able to increase HIF-1α protein but not mRNA expression. In hypoxic condition, Id-1 increased VEGF expression by stabilizing HIF-1α protein. By anti-sense approach, Id-1 downregulation led to suppression of HIF-1α-mediated VEGF production, and resulted in reduced tumor growth and vascularity when the HCC cells were injected subcutaneously in nude mice. CONCLUSION: Id-1 promoted angiogenesis in HCC through stabilization of HIF-1α protein and increased VEGF expression. Downregulation of Id-1 may be a novel target to inhibit angiogenesis in HCC. ©American Association for Cancer Research